Background and Objectives: The key pathogenetic mechanism of glucose metabolism disorders, insulin resistance (IR), can be assessed using the Homeostasis Model Assessment of IR (HOMA-IR). However, its application in clinical practice is limited due to the absence of cut-offs. In this study, we aimed to define the cut-offs for the Czech population. Methods: After undergoing anthropometric and biochemical studies, the sample of 3539 individuals was divided into either nondiabetics, including both subjects with normal glucose tolerance  and prediabetics (n = 1459), or diabetics (n = 133). The optimal HOMA-IR cut-offs between subgroups were determined to maximize the sum of the sensitivity and specificity for diagnosing type 2 diabetes mellitus (T2DM) or prediabetes. The predictive accuracy was illustrated using receiver operating characteristic (ROC) curves. Logistic regression was performed to assess the association between a target variable (presence/absence of T2DM) depending on the HOMA-IR score as well as on the age and sex. Results: The HOMA-IR cut-off between nondiabetics and diabetics for both sexes together was 3.63, with a sensitivity of 0.56 and a specificity of 0.86. The area under the ROC curve was 0.73 for T2DM diagnosing in both sexes. The HOMA-IR cut-off between the NGT subjects and prediabetics was 1.82, with a sensitivity of 0.60 and a specificity of 0.66. Logistic regression showed that increased HOMA-IR is a risk factor for the presence of T2DM (odds ratio (OR) 1.2, 95% confidence interval (CI) 1.14–1.28, p < 0.0001). The predictive ability of HOMA-IR in diagnosing T2DM is statistically significantly lower in females . The results are valid for middle-aged European adults. Conclusions: The results suggest the existence of HOMA-IR cut-offs signaling established IR. Introduction of the instrument into common clinical practice, together with the known cut-offs, may contribute to preventing T2DM. The global burden of type 2 diabetes mellitus (T2DM) is expected to double by 2030, with impacts on both the health systems and economies of affected countries. There are growing numbers of diabetics not only in affluent countries but also in low- and middle-income countries . T2DM haT2DM is a disease state with distortions in the relationship between circulating glucose and insulin values in a manner that does not reflect the systemic insulin sensitivity. Hyperglycemia is the hallmark of T2DM and is accompanied by “glucose toxicity” with respect to insulin secretion. Consequently, studies assessing the relationship between indices based on fasting plasma glucose (FPG) and insulin levels and clamp measures could reflect falsely inflated slopes and correlation coefficients in regression equations when data from nondiabetic and diabetic subjects are included in the same regression analyses . That isThe present study defines HOMA-IR cut-offs for middle-aged male and female in the Czech Republic.n = 1947), prediabetics (n = 1459) and diabetics (n = 133). The NGT subjects together with the prediabetics formed a subgroup of nondiabetics, which was used for the HOMA-IR cut-off analysis. The prediabetics were individuals with at least one of the following conditions: (1) FPG 5.6–6.9 mmol/L; (2) 120-minute plasma glucose 7.8–11.0 mmol/L after a 75 g oral glucose tolerance test (OGTT). The diabetics were exclusively individuals with T2DM, which was defined as (1) FPG ≥ 7.0 mmol/L; or (2) 120-minute plasma glucose ≥11.1 mmol/L after OGTT. Most of the diabetics received long-term therapy with oral antidiabetic drugs, most frequently metformin. Insulin therapy was an exclusion criterion as well as very high FPG which would limit the use of the HOMA-IR. Individuals in the NGT subgroup included the other patients attending the center with normal glucose metabolism.The sample comprised 3539 middle-aged individuals examined in the co-author’s internal medicine center in Hradec Králové who agreed to participate. Data were collected in 2009–2015. The sample was divided into three subgroups: subjects with normal glucose tolerance . Insulin was determined by the Chemiluminescent Microparticle Immunoassay method on Architect i1000SR . All analyses were performed according to the manufacturer’s instructions and after verification of methods.The HOMA-IR was calculated with the following formula  :HOMA-IRp < 0.05. A confusion matrix comparing the actual presence of the diagnosis of T2DM against its prediction was performed. A statistical analysis was performed using the computing environment R . The Wilcoxon signed-rank test  was used to compare numerical characteristics between the given subgroups. For each sex, cut-offs were determined so that the sum of sensitivity and specificity was maximized. Sensitivity and specificity were considered equally important and, therefore, their relative weights in the sum were 1:1. In other words, we searched for the maximum of Youden’s index as a function of sensitivity and specificity . The accThe basic characteristics of all subjects in the three subgroups are shown in p < 0.0001). For HOMA-IR, a statistically significant predictor is sex; when HOMA-IR scores are used to estimate the odds of T2DM, with the other variables remaining unchanged, the odds of T2DM are 0.6567-fold higher in females compared to males (p = 0.0384). Another statistically significant predictor is age, but with OR close to one (p < 0.0001). The accuracy of the prediction for the logistic regression model with the HOMA-IR predictor is 0.962 to 0.963 (p < 0.0001) according to the confusion matrix.p < 0.0001) according to the confusion matrix.The results suggest the existence of HOMA-IR cut-offs signaling established IR. This condition is one of the main pathogenetic mechanisms of T2DM, atherosclerosis and metabolic syndrome which increasingly contribute to the morbidity and mortality of the affluent populations . The maiOne of the factors potentially influencing insulin sensitivity is the increased resting metabolic rate, as shown in a large cohort of 782 adults . It has IR and associated metabolic abnormalities are increasingly being reported in the adolescent population. The HOMA-IR cut-off as an indicator of metabolic syndrome in adolescents has not been established. In Indian adolescents, HOMA-IR scores increased with sexual maturity and with progression from normal to obese. A HOMA-IR cut-off of 2.5 provided the maximum sensitivity and specificity in diagnosing . ObesityHOMA-IR is a robust instrument currently used mostly in large population studies to identify IR already present in prediabetes. Prediabetes is a high-risk state for T2DM, especially in patients who remain with prediabetes despite intensive lifestyle intervention. Reversion to normal glucose regulation, even if transient, is associated with a significantly reduced risk of future T2DM . It is, The study has its limitations. In all subjects, the HOMA-IR scores were calculated based on the single measurements of blood glucose and insulin levels at a single time point; these may change over a short period of time. The diabetic subgroup for identification of the cut-off is much smaller than without the diabetes subgroup. The euglycemic clamp to confirm the presence of IR was not performed. The validity of the HOMA-IR should be carefully considered in subjects with a lower BMI, lower beta cell function, and high FPG such as in T2DM with the insulin secretory defect . HoweverGiven the current global epidemic of obesity, it is rather desirable to prevent T2DM. Using data on a group of individuals collected over a period of six years, the HOMA-IR cut-off of 3.63 was determined for the middle-aged Czech population. These may identify patients with already established IR at the stage of T2DM. However, the sensitivity of the cut-offs was 0.56. Therefore, this test cannot currently be recommended as a reliable tool for IR screening. The specificity of the test (0.86) confirms its good validity. The HOMA-IR cut-off between subjects with NGT and prediabetics was 1.82 , and thus prediabetes is bordered with the HOMA-IR values of 1.82 and 3.63. The determination of the optimal cut-off for diagnosing at-risk individuals in common practice is beneficial for early detection of prediabetes.


Further, the flush-like temperature increases were strongly attenuated in ovariectomized mice lacking αCGRP due to a genetic modification. Collectively, our findings suggest that CGRP is an important mediator of experimentally induced hot flushes and they identify CGRP antagonists as promising treatment candidates for women and possibly also men with hot flushes.Hot flushes are common and troublesome symptoms of menopause. The neuropeptide calcitonin gene-related peptide (CGRP) is increased in plasma during hot flushes but it has not been clear if CGRP is causally involved in the mechanism underpinning the flushes. Here, we examined the effect of interventions with CGRP in a mouse model of hot flushes based on flush-like temperature increases triggered by forced physical activity in ovariectomized mice. Compared to normal mice, ovariectomized mice reacted with an exaggerated, flush-like, temperature increase after physical exercise. This increase was completely blocked by the non-peptide CGRP-antagonist MK-8825 (-0.41 degrees Celsius, 95% CI: -0,83 to 0,012,  Hot flushes are classic menopausal symptoms in women  but are The neuropeptide calcitonin gene-related peptide (CGRP), which is found predominantly in sensory C and Aδ nerve fibers, has been suggested to be involved in hot flushes . CGRP isDespite the correlational data linking CGRP and hot flushes, no attempts have been made to determine if blocking the actions of CGRP inhibits flushes. The pharmacological tools are available; antagonists toward CGRP-receptors have been developed and tested in human phase II and phase III studies, mainly for treatment of migraine  and MK-8RRID:MMRRC_036773-UNC) and were on a C57Bl/6 background and weighed 18–26g  female mice weighing 25–30 g were used. The mice lacking αCGRP and their littermate controls were from MMRRC  of oxygen/nitrous oxide at day 1. Beginning on day 15, a program was started in which mice were trained to run on a motor-driven treadmill  for 3 weeks. During the first week mice were allowed to run at 4 m/min for 1 min and then 7 m/min for 5 min each once a week. During the second week, mice were allowed to run at 7 m/min for 5 min and then 15 m/min for 5 min each once a week. During the third week mice ran at 15 m/min for 10 min twice a week. Thirty-five days after the ovariectomy, mice were subjected to treatments and measurement. Mice that ran less than 10 min or were not willing to go inside the mouse-holder were excluded from the experiment. An overview of the experimental design is presented in Figure The ovariectomized mice were randomly allocated to treatment with vehicle  or the CGRP-antagonist MK-8825 . Twenty minutes before running, 200 μl of sodium chloride  or CGRP-antagonist MK-8825 at a dose of either 10 mg/kg body weight or 50 mg/kg body weight of (8750 μg/ml in 0.9% NaCl) was injected intraperitoneally.Tail-skin temperature was measured as previously described [28]. In brief, mice were restrained in a holder and tail-skin temperature was measured at the dorsal surface of the tail one cm from its base with a thermotracer  for 15 min, 30 min before exercise on the treadmill. The mean tail-skin temperature during the first 6-min period was defined as basal tail-skin temperature. The mice were then injected with either saline or MK-8825 (50 mg/kg of body weight) and, 20 min after the intraperitoneal injection, allowed to run for 10 min (15 m/min) on a motor-driven treadmill . Tail-skin temperature was measured for 15 min after the exercise, and changes in tail-skin temperature at 1–6 min after exercise were assessed. The change in tail-skin temperature was calculated by subtracting the basal tail-skin temperature (see above) from the mean tail-skin temperature after exercise. All measurements of tail-skin temperature were performed between 9:00 and 12:00 h and the room temperature was maintained at 22–25°C throughout the measurement period.Mice were placed in an open-field arena (45 cm x 45 cm), and their activity was recorded for 15 min with a video camera placed above the arena. EthoVision tracking software  was used for data processing and analysis. Total distance traveled, and the time mice spent in the center of the arena was quantified.®Gold Antifade (ThermoFisher). The images were obtained on fluorescent microscope (Nikon Eclipse E800).Mice were perfused with saline followed by 4% paraformaldehyde in PBS. The tissue was postfixed in the same solution for 4 h in 4°C and placed in 30% sucrose till saturated. 40 μm free floating sections were blocked in 10% Normal Donkey Serum in PBST  for 24 h. After blocking, sections were incubated in a mixture of primary antibodies  over night. The next day sections were rinsed 5 times with PBST and moved to a mixture of secondary antibodies: goat anti-chicken Alexa Fluor 488, 1:1000 and donkey anti-rabbit Alexa Fluor 568, 1:1000. After 2 h the sections were rinsed in PBST and mounted in ProLongT-test was used when comparing two groups and 1-way ANOVA followed by Holm-Sidaks multiple comparisons test was used when comparing more than two groups. Differences at the p < 0.05 level were considered significant.All data were represented as the mean ± SEM. Students n = 6) compared to mice subjected to sham-surgery   compared to control mice  (p < 0.0001)  (p < 0.0001) . A lower dose of MK-8825 (10 mg/kg) did not significantly reduce the increase in tails skin temperature in ovariectomized mice  (p = 0.26). Compared to whereas the high dose of MK-8825 blocked the flush-like events, it should be noted that such an effect could reflect an inability to mount a response due to unspecific effects on the well-being of the mice. To examine if the mice were negatively affected in an unspecific way by the drug, we examined locomotor activity after the two doses of MK-8825. Mice treated with MK-8825 at a dose of 50 mg/kg did not display any obvious differences in locomotor activity  compared to Ovx mice given saline  (p = 0.90)   Figure . Next, w) Figure . In cont) Figure . Compare) Figure , which s) Figure , which dn = 4)  (p = 0.0031)  Figure . This in) Figure .In this study, we found that administration of the CGRP receptor antagonist MK-8825 and deletion of αCGRP block or attenuate the exaggerated tail-skin temperature increases observed in ovariectomized mice after exercise. These findings complement earlier studies showing increased levels of CGRP in plasma during flushes in humans .Our findings clearly show that CGRP is important for hot flushes in a mouse model. However, the way by which CGRP is involved in the mechanisms underpinning hot flushes is still not clear. The fact that CGRP is a potent vasodilator and sweat gland activator, leads to flush-like symptoms when injected, and that it increases acutely in plasma during hot flushes may suggest that it is involved in the efferent link of the flush, i.e., that CGRP comes into play after the hypothalamus has initiated the flush-response . Since CAnimal models of hot flushes have proved difficult to develop, but measurements of tail skin temperature in ovariectomized mice is an attractive model since it mirrors the peripheral vascular events seen in hot flushes (vasodilation in the skin leading to an increased temperature) and these effects can be blocked by estrogen supplementation . The modDW, DE, ET, MH, and A-CS conceived the study. HD, HB, DW, AS, JZ, and EP performed the experiments. A-CS acquired the drug. DE supervised all animal experiments. DW, DE, and A-CS drafted the first version of the manuscript. All authors contributed substantially to the data analyses, revision of the manuscript and approved the final submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aim: As high gamma-glutamyltransferase level or low albumin had negative impacts on the prognosis of hepatocellular carcinoma (HCC), the prognostic role of albumin to gamma-glutamyltransferase ratio (AGR) in HCC patients after hepatectomy remains unclear.Methods: Between January 2007 and December 2015, 1143 HCC patients after hepatectomy were reviewed from a prospectively maintained database in West China Hospital. All qualified patients (n = 959) were classified as training set  and validation set . A time-dependent receiver operating characteristic (ROC) curve analysis was performed to evaluate the performance.Result: AGR = 0.5 was identified as the best cut-off point to predict recurrence free survival (RFS) and overall survival (OS) in the training set. Low AGR was related to poor tumor characteristics and high systemic inflammation. Based on the multivariate analysis, high AGR was an independent predictor for better RFS and OS with an hazard ratio of 0.696 and 0.673. The high AGR group had better RFS and OS than the low AGR group in the training set as well as the validation set. The AGR-based score (AGR-PLR) could stratify HCC patients into three subgroups with different prognosis in the training and validation set. Patients with score 1 had a worse prognosis than those with AGR-PLR score 0, but better than those with AGR-PLR score 2. The predictive accuracy of the AGR-PLR score appeared superior to that of the AGR or PLR alone.Conclusions: we firstly reported that AGR ≤ 0.5 was an independently prognostic factor in HCC after hepatectomy. The AGR-PLR score could further improve the discriminatory ability of prognosis. Hepatocellular carcinoma (HCC) is one of most common malignancies worldwide, with an increasing incidence rate. Particularly, China accounts for 51% of the deaths from liver cancer worldwide because of the high prevalence of hepatitis B viral (HBV) infection . CurativIncreasing evidences indicated elevated serum gamma-glutamyltransferase (GGT), an enzyme locating at the surface of the epithelial cells, which was related to inflammation and carcinogenesis. Abnormal GGT was a prognostic predictor for poor overall survival (OS) of HCC patients after transcatheter arterial chemoembolization (TACE)  or radioIn this study, we aimed to assess the prognostic role of AGR in HCC patients after radical hepatectomy. Furthermore, we would compare the predictive power between the novel AGR and other inflammatory scores for HCC patients after hepatectomy.Between January 2007 and December 2014, the data of 1,143 HCC patients who underwent hepatectomy were collected from the Department of Liver Surgery and Liver Transplantation Center in West China Hospital. Our inclusion criteria were as follows: (1) pathologically proven HCC; (2) first hepatectomy; (3) Child–Pugh grade A or Child–Pugh B. Exclusion criteria: (1) with major vascular invasion; (2) with positive surgical margin; (3) patients with recurrent HCC; (4) incomplete clinicopathological information or follow-up data. Finally, 959 patients were included in the current study. Patients between June 2007 and October 2012 were defined as the training set and patients between November 2012 to March 2015 were defined as the validation set . The fol2 test with the Yates correction or Fisher's exact test. Continuous variables (presented as the mean ± SD) were compared using analysis of Student's t-test or the non-parametric Mann-Whitney test . The optimal cutoff value of AGR, PLR and NLR was determined by X-tile software (https://x-tile.software.informer.com/). The risk factors associated with prognosis of patients with HCC were evaluated using Cox regression analysis. Kaplan–Meier analysis and the log-rank test were performed by comparing the differences of the cumulative survival of HCC patients between groups. The discriminatory ability of AGR and PLR were evaluated by time-dependent area under receiver operating characteristic curve (AUROC). Time-dependent ROC was depicted using Kaplan-Meier method via the survival ROC package in R. The statistical analyses were performed using the SPSS 20.0 software  and R project version 3.4.1. A P-value <0.05 in two-tailed tests was considered as statistically significant.Frequency was compared between groups using the χn = 480) and the validation set (n = 479). In the training set, we adopted X-tile software to confirm that the optimal cutoff point for AGR was 0.5 by minimum P-value from log-rank χ2 test, based on which patients were divided into two groups with the strongest discriminatory capacity ; higher rate of patients with multiple tumors (P = 0.011); higher rate of the presence of MVI (P = 0.004); and satellite lesions (P = 0.001); higher Child-Pugh grade (P = 0.013); elevated ALT and AST (P < 0.001); higher NLR and PLR level (p = 0.036 and p = 0.013); and higher INR and Fibrinogen (p = 0.025 and p < 0.001). There were no significant differences about other variables between both groups . In order to reduce the possible significant interactions among them, we selected significant variables in the univariate analysis and included them into the Cox proportional hazards model. We found that positive HBeAg , tumor number, MVI , satellite lesions, and PLR  were independent risk factors for RFS, while a higher AGR  was associated with decreased risk of RFS . In contrast, tumor size , tumor number , MVI , satellite lesions , tumor differentiation , and PLR  were associated with an increased risk of OS . The 1-, 3-, and 5-year OS rates were 94.7, 79.3, and 64.4% for the high-AGR group, and 83.1, 52.4, and 39.3% for the low-AGR group, respectively (p < 0.001). There was statistically significant in the prognosis of HCC patients . Meanwhile, the high-AGR group had a better OS   .Since the albumin in the AGR was affected by liver function, we made the survival analysis in the patients with liver cirrhosis. Interestingly, the high-AGR remained to be associated with better prognosis in term of RFS and OS .As was shown in the multivariate analysis, high PLR as an inflammatory score was inversely correlated with RFS and OS , 3. HCC p < 0.001, score 1 vs. score 2:p = 0.062), Similarly, Patients with AGR-PLR score 1 had better 1-, 3-, and 5-year OS rates than those with AGR-PLR score 2, but worse than those with AGR-PLR score 0 .We allocated 1 point for AGR ≤ 0.5 or PLR > 167.7. The AGR-PLR score consisting of 0, 1, and 2 was built in the training set. There were statistical differences in RFS and OS among the three groups divided according to AGR-PLR score . Patientp = 0.020, score 1 vs. score 2: p = 0.002) and OS   . Based o= 0.022) .In China, HBV infection was the most common cause of HCC . HepatitIn recent years, the host inflammatory response caught much attention in tumor progression or metastasis . AGR wasIn the current study, we identified that the optimal cutoff value of AGR was 0.5 by using the X-tile software with the strongest discriminatory ability. We found that low AGR (≤0.5), as a reflection of low albumin or high GGT, was related to poor liver function (higher Child-Pugh grade), active underlying inflammation (elevated ALT and AST), high systemic inflammation (higher PLR and NLR), and aggressive tumor characteristics . In order to adjust the effect of other potential risk factors on the AGR, we further adopted the multivariate analysis. We found that AGR > 0.5 was the only favorable factor associated with HCC recurrence and long-term survival in the training set. Based on the survival analysis, patients with AGR> 0.5 had 5-year of 64.4%, while patients with AGR ≤ 0.5 had 5-year of 39.3%. Similarly, in the validation set, we demonstrated that AGR > 0.5 could discriminated a subgroup of HCC patients with better RFS and OS.We also investigated the role of two other inflammatory score, namely NLR and PLR. The cutoff value of the inflammatory score were 3.1 and 167.7 using the same method. Consistent with other studies , 31, theThere were some limitations in the current study. Firstly, this was a single-center and retrospective study. In order to reduce the selection bias, we stratified patients into a training set and validation set based on different time spans. Secondly, the presence of major vascular invasion had the strongest negative impacts on the prognosis of HCC patients. It might cover some risk factors. In the current study, we excluded the patients with macro-vascular HCC. Thirdly, some factors differed for OS and RFS in the multivariate analyses because it resulted from the different endpoints. However, the majority of factors affecting the OS and RFS remained the same. Finally, since almost patients included in the study had HBV infection, its prognostic role in HCC with hepatitis C or no infection of hepatitis virus might differ.In conclusion, we provided strong evidence that the AGR could offer prognostic information on prognosis of patients with HCC after hepatectomy. In addition, we confirmed that AGR-based score (AGR-PLR score) might further stratify the HCC patients with different prognosis.TW proposed the study. JS, LT, and CL performed the research. JS, XZ, and GL collected and analyzed the data. JS wrote the first draft. XZ, WP, TW, and JY reviewed the manuscript. All authors contributed to the interpretation of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The GNS@m-SiO2 nanoparticles are further encapsulated within a thermo-responsive poly(N-isopropylacrylamide-co-acrylic acid) hydrogel (PNIPAM-co-AA). The multi-responsive composite NPs were designed to create thermally and optically modulated drug-delivery vehicles with a m-SiO2 layer providing additional non-collapsible space for drug storage. The influence of the m-SiO2 layer on the efficacy of loading and release of methylene blue, which serves as a model for a small-molecule therapeutic drug, was evaluated. The “smart” core@shell composite NPs having a m-SiO2 layer demonstrated an improved capacity to load and release small molecules compared to the corresponding NPs with no m-SiO2 shell. Additionally, an efficient response by the composite NPs was successfully induced by the thermal energy generated from the gold nanoshell core upon exposure to near infrared (NIR) stimulation.A “smart” core@shell composite nanoparticle (NP) having dual-response mechanisms  was synthesized, and its efficacy in the loading and release of small molecules was explored. These core@shell NPs are composed of an optically active gold nanoshell (GNS) core and a mesoporous ( Motivation for the synthesis of metal nanoparticles (NPs) and their composites has been driven in recent years by the unique and tunable optical properties of these materials and their potential utility in various applications. However, simple metal nanoparticles offer limited utility in many practical applications due to the narrow breadth of their optical properties. This drawback has led to the exploration of alternative nanoparticle configurations, particularly core@shell geometries. Among the many core@shell composite particles, those that respond to stimulation from the core particle  and the shell through an independent or correlated external stimulus  have garnered interest. Numerous studies have reported their fabrication and potential applications in controlled drug delivery ,2, chemi2)—optically transparent and easy to synthesize/modify—and a biocompatible gold shell prove beneficial [For biological applications such as DDSs, core@shell composite materials with a dielectric core, such as silica . To overcome this drawback, the growth of a stimuli-responsive hydrogel polymer coating on the m-SiO2 layer can be incorporated due to its ability to release model drugs upon the collapse of the hydrogel matrix [Another aspect that needs consideration for the development of a targeted photothermally-exposed drug-delivery system is the selection of an optimal shell material. The shell material should be one that is biocompatible and have the capability of acting as a “smart” delivery vehicle. The drug-carrying capacity of  2-50 nm ,26. Howel matrix ,28.Hydrogel polymers have proven to be popular materials for a variety of technological applications such as drug-delivery systems, chemical separations, and catalysis ,4,29,30.m-SiO2-coated NP system that responds to external stimuli to both load and release therapeutic agents. Herein, we present a method for the synthesis of discrete NIR-responsive and temperature-responsive hydrogel-encapsulated m-SiO2 gold nanoshells (GNS@m-SiO2@hydrogel) and their use in the loading and release of methylene blue, which serves as a model for a small-molecule therapeutic drug. The hydrogel layer used in our system was a poly(N-isopropylacrylamide)-co-acrylic acid, P(NIPAM-co-AA), consisting of 6 wt% acrylic acid and having an LCST of 34-40 °C. In particular, we wished to use the m-SiO2 layer to prevent unwanted structural changes to the gold shell during photomodulation and to act as a reservoir for carrying drug payloads to targeted locations. An analogous GNS-based system having no m-SiO2 layer was also evaluated to evaluate the performance of our composite system. Taken as a whole, this approach of forming composite nanoparticles involving a core nanomaterial coated with a m-SiO2 layer and encapsulated by a hydrogel modulated by an external stimulant offers a promising new system for nanomedicinal therapeutics.An ongoing goal of our research is to develop a versatile hydrogel-encapsulated-2@hydrogel and GNS@m-SiO2@hydrogel is shown in 2 coating was further modified through base-etching using a procedure reported by Yin et al. [m-silica layer with the hydrogel copolymer, the surface was modified using 3-(trimethylsilyl) propylmethacrylate (MPS). To encapsulate the composite NPs with the thermo-responsive hydrogel copolymer NIPAM-co-AA, emulsion polymerization was used [The strategy used to synthesize the GNS@SiOn et al. . To coatwas used ,42,43. Tm-silica-coated nanoparticles was found to be convenient and reliable due to the high solubility of the starting materials under optimized reaction conditions. Moreover, there was no need for purification steps other than filtration. Phase separation in some instances was observed under various conditions, such as reaction time, temperature, or the concentration of the monomers. However, using the procedures outlined above, we found that we could reliably and reproducibly fabricate hydrogel-encapsulated m-silica-coated gold nanoshells. Further, the composite particles were found to be colloidally stable, resisting aggregation for more than two months at room temperature.Using gold nanoshells as core materials allows for strong optical extinctions that can be precisely tuned anywhere from ~600–1200 nm, giving rise to composite particles that can be activated for drug release using light at NIR wavelengths . Gold na2), m-SiO2-coated gold nanoshells (GNS@m-SiO2), and hydrogel-encapsulated m-SiO2-coated gold nanoshells (GNS@m-SiO2@hydrogel), depicting changes associated with each step in the synthesis. The individual images of each step of the synthetic procedure provide a means of determining from one image to the next which part of the composite image is associated with each modification step. The transmission electron microscope (TEM) images confirm the results obtained by scanning electron microscope (SEM) and are shown as insets in 2 coating exhibited a thickness of ~90 nm. Furthermore, etching of the silica shell, to obtain the m-SiO2, led to no change in the thickness of the shell; the diameter of the particles remained at ~240 nm before , possible defects in the gold shells , the presence of a small number of incomplete gold shells, and/or the m-silica layer (~50 nm) on the gold nanoshell. EDX elemental data provide evidence that the key structural components are present for the hydrogel-encapsulated m-SiO2-coated GNSs. Moreover, the EDX analysis suggests the presence of silica, Au, and the hydrogel copolymer in the final composite nanoparticle and, indirectly supports the hydrogel copolymer as the dominant material on the surface of the composite NPs.The final composite NPs were analyzed with energy dispersive X-ray spectroscopy (EDX) to confirm their elemental composition see . Analysi2, GNS@m-SiO2, and GNS@m-SiO2@hydrogel are provided in max) at 530–570 nm. However, gold nanoshells have been shown to exhibit a broad plasmon band in the 600–1500 nm range [m-SiO2@hydrogel NPs have a peak position similar to that of the unmodified gold nanoshells due to the thinness of the overlying layers. However, the GNS@m-SiO2@hydrogel NPs also exhibit a scattering pattern at shorter wavelengths (300–600 nm). We consider the differences in λmax to be minimal among the GNS@m-SiO2@hydrogel, GNS@SiO2, GNS@m-SiO2, and the bare gold nanoshells. The minimal difference would be due to changes in the refractive index of the hydrogel, m-SiO2, or SiO2 layers which lead to a slight increase in ultraviolet (UV) scattering at shorter wavelengths (300–600 nm), a minor red-shift by the SiO2 and hydrogel layers, and a blue shift associated with the m-SiO2 layer. There is, however, no distinctive shift for λmax for the GNS@m-SiO2@hydrogel as compared to the bare gold nanoshells.The optical properties of the GNS, GNS@SiOnm range . The bronm range . The finThe disruption of hydrogen bonds between water and hydrophilic sites within the hydrogel polymer backbone (such as -C=O and -NH) above the LCST has been shown to lead to the breakdown of hydrogel polymer matrices through reduced internal electrostatic repulsion, giving rise to the collapse of the hydrogel polymer layer . Convers2, GNS@SiO2@hydrogel, and GNS@m-SiO2@hydrogel at rt (25 °C) and 40 °C, shown in 2 (~300 nm) remains relatively constant with changes in temperature, while the diameter of the GNS@m-SiO2@hydrogel NPs decrease  with increasing temperature and increase (~570 nm) with decreasing temperature; a similar trend is also observed with the GNS@SiO2@hydrogel NPs  was used to record the hydrodynamic diameter changes of the GNS@SiOogel NPs . Note, bm-SiO2 interlayer and hydrogel layer, can respond rapidly and efficiently to changes in temperature slightly above that found in the body (vide supra). The localized increase in temperature can give rise to the release of small molecules from the hydrogel matrix, providing a targeted drug-delivery system.Light at wavelengths between 700 and 1200 nm can penetrate human skin without causing any damage . Moreovem-SiO2@hydrogel at a constant temperature of 25 °C and 40 °C as shown in m-SiO2 coating provides additional space for carrying small molecules due to their large pore volume (ca. 1 cm3/g) and tunable mesopore diameter [To evaluate the maximum amount of molecular loading and delivery of the GNS@diameter . By mixi2 on MB loading efficiencies by comparing the GNS@m-SiO2@hydrogel to the analogous GNS-based composite particle without the mesopores, GNS@SiO2@hydrogel. With respect to the concentration of MB, the loading efficiencies were determined to be ~61 % and ~71 %, for the GNS@m-SiO2@hydrogel and GNS@SiO2@hydrogel, respectively, as shown in m-SiO2@hydrogel is ~10 % higher than that for the GNS@SiO2@hydrogel due to the higher surface area of the silica shell coming from the large pore volume of the mesoporous silica interlayer [We first determined the influence of the mesoporous SiOterlayer ,26.m-SiO2@hydrogel at a constant temperature of 40 °C as compared to the GNS@SiO2@hydrogel at 40 °C, as shown in After loading, the MB-loaded hydrogel-coated composite NPs were collected and redispersed in Tris buffer to evaluate their release profile. We then maintained our samples at a constant temperature of 40 °C, which is just above the LCST of our P(NIPAM-co-AA) hydrogel. The samples were analyzed every 24 h for 144 h. At the end of each 24 h timeframe, the MB-loaded hydrogel-coated composite NPs were centrifuged, and the concentration of the released MB in the supernatant was determined by UV-vis spectroscopy . As a control, we performed the same experiment at a constant temperature of 25 °C, which is ~10 °C lower than the LCST. The cumulative release of MB was evaluated at selected intervals of time, and the collected data are shown in m-SiO2@hydrogel with MB as described above . The composite NPs were exposed to a NIR laser (wavelength = 810 nm) for 144 h. The composite NPs were then centrifuged at selected intervals during the laser exposure protocol , nitric acid, and hydrochloric acid were all from EM Science, Hatfield, PA, USA, while potassium carbonate was from Aldrich, St. Louis, MO, USA. 3-(Trimethylsilyl)propylmethacrylate , N-isopropylacrylamide , acrylic acid , and the cross-linker N,N′-methylenebisacrylamide  were obtained from Acros; tetraethylorthosilicate (TEOS), tetrakis(hydroxymethyl)phosphonium chloride (THPC), polyvinylpyrolidone , and 3-aminopropyltrimethoxysilane (APTMS) were purchased from Aldrich, St. Louis, MO, USA; and, hydrogen tetrachloroaurate(III) hydrate came from Strem, Newburyport, MA, USA. Water was purified to a resistance of 18 MΩ  and filtered using a 0.22 μm filter to remove any impurities. All glassware and equipment used in the experiment were first cleaned in an aqua regia solution , then cleaned in a base bath  and lastly, rinsed with Milli-Q water prior to use.The chemicals used in this study were purchased from the following companies: ethanol, formaldehyde, sodium hydroxide, ammonium persulfate , ammonium hydroxide  was quickly added. The color of the solution changed from colorless to reddish brown at once. The solution was stored in the refrigerator at 4 °C for at least three days to age. The size of our aged-THPC gold seed particles was ~2–3 nm in diameter. We deposited the aged-THPC gold seed particles onto the silica core particles using a modified method by Westscott et al. [A reduced gold (THPC gold) solution was prepared by using a modified procedure found in the literature ,56. A 1.t et al. . Briefly2CO3 and HAuCl4·3H2O. An aliquot of K2CO3  was completely dissolved in 100 mL of Milli-Q water by stirring for 10 min, and then 2.0 mL of 1 wt% HAuCl4·3H2O was added to the mixture. The color of the solution transformed from yellow to colorless within 30–40 min. After preparation, the K-gold solution was kept in the refrigerator at 4 °C for one day under dark conditions. To grow the gold shell on the THPC gold-seeded silica nanoparticles [We prepared an aqueous solution for growing the gold shells (K-gold solution) using Karticles , 8 mL of2-coated composite nanoparticles [2-encapsulated gold nanoshells, which were isolated by washing with copious amounts of ethanol before redispersing in water.Polyvinylpyrolidone (PVP) was employed to induce phase transfer of the nanoshells from water to ethanol and to stabilize the surface of the gold shells to obtain SiOarticles ,39. The 2 layer was turned into a porous structure by using the procedure reported by Yin et al. [m-SiO2 coated particles were suspended in Milli-Q water.The surface of the SiOn et al. . More spm-SiO2-coated nanoparticles were prepared by an emulsion polymerization procedure in an aqueous solution. To afford the hydrogel encapsulation, the surfaces of the m-SiO2-coated particles GNS were initially functionalized with vinyl groups. To achieve this, a suspension of the m-SiO2-coated particle was mixed with MPS (0.20 mL) under vigorous stirring. The reaction mixture was stirred for 24 h at room temperature (rt). Subsequently, the temperature was elevated to 85 °C in order to improve the bond affinity between MPS and the m-SiO2 shell. The resultant particles were separated and washed with ethanol several times before redispersing in 10.0 mL of ethanol. An aqueous solution containing NIPAM, AA, and BIS, was mixed with the MPS-modified m-SiO2-coated NP ethanolic solution (5 mL) and placed in a three-necked round-bottomed flask equipped with a reflux condenser and an inlet for argon gas. The solution was degassed with argon for 1 h to remove any oxygen in the flask. This step was performed to reduce the presence of oxygen in the solution, because oxygen can intercept radicals and disrupt the polymerization process. NIPAM , AA , and the cross-linker BIS  were added to the mixture and stirred for 15 min. The mixture was then heated to 70 °C in an oil bath followed by addition of the initiator APS  to induce the polymerization. The reaction was allowed to proceed for 7~8 h followed by cooling the solution to rt. The final particles were washed with deionized water and filtered with a 1 μm membrane to remove any micron-sized impurities and/or any aggregated particles at the end of the reaction. The filtered solution was centrifuged at 30 °C for 1 h at 5000 rpm two times, and the supernatant was separated to remove any unreacted materials and soluble side products. The purified nanoparticles were then diluted with Milli-Q water and stored at rt for later analysis.The hydrogel-encapsulated m-SiO2@hydrogel NPs and GNS@SiO2@hydrogel NPs were ~550 nm in diameter, a size that was produced by controlling the amount of monomer, the amount of initiator, and the reaction time. A thicker coating of the hydrogel on the gold nanoshells was also achieved by increasing the concentration of the monomers, but this thicker coating was less reproducible than the thin coating. We also observed the formation of multiple m-SiO2-coated gold nanoshell cores within a single hydrogel shell in the thick hydrogel-coated samples. Therefore, we chose to focus on analyzing the hydrogel-coated gold nanoshells having ~550 nm diameters.The size of the GNS@2@hydrogel NPs as a representative example. A 20 mL aliquot of the GNS@SiO2@hydrogel solution was placed under vacuum to form a white powder followed by addition of 5 mL of a solution of 0.00002 M MB in Tris buffer. After stirring overnight at rt, the MB-loaded GNS@SiO2@hydrogel NPs were separated from the excess MB molecules by centrifugation at 5000 rpm for 30 min, 2–3 times. The MB-loaded hydrogel-coated composite NPs were deposited as a residue on the bottom of the centrifuge tube after decanting the solution of excess MB. For quantification of the MB loaded inside the hydrogel-coated composite NPs, the UV-vis absorption spectrum of the supernatant MB solution was recorded. The amount of the excess MB was calculated using calibration curves generated at λ = 664.5 nm from standard solutions prepared at known MB concentrations. By subtracting the calculated MB from the original MB solution, the amount of MB loaded inside the hydrogel was obtained.To load the MB dye inside the hydrogel of the hydrogel-coated nanoparticles, a dye loading procedure was developed and is described as follows using the GNS@SiOFor the release study, 5 mL of Tris buffer was added to the centrifuge tube containing the MB-loaded hydrogel-coated composite NPs. The solution was stirred for selected intervals of time separately at 40 °C, at 25 °C, or with NIR laser exposure (wavelength = 810 nm) and then centrifuged as described above. The supernatant was collected by decantation, and the concentration of MB in the supernatant was recorded by UV-vis spectroscopy to calculate the released amount of MB from the hydrogel-coated composite NPs. The procedure was repeated for each time interval (every 24 h for 144 h) to generate the release profiles shown in the Analysis by SEM was carried out using a LEO-1525 scanning electron microscope operating at 15 KV. To collect FE-SEM images, synthesized nanoparticles were deposited on a silicon wafer and completely dried at rt overnight prior to analysis. All the samples were examined at magnifications of 10 K, 30 K or 50 K, and 100 K in order to demonstrate the overall uniformity of the particles.Analysis by TEM was carried out using a JEM-2000 FX transmission electron microscope (JEOL) equipped with elemental analysis by EDX  at an accelerating voltage of 200 kV. All of the TEM samples were deposited on 300 mesh Holey carbon-coated copper grids and dried before examination.2 particles, bare GNS, m-SiO2-coated GNS and GNS@m-SiO2@hydrogel NPs. UV-vis spectra of the prepared gold nanoshells were collected by diluting the samples with Milli-Q water, transferring them to a quartz cell, and scanning over a range of wavelengths (250–1000 nm). The GNS@m-SiO2@hydrogel NPs were analyzed as prepared without dilution. The spectra of distinct batches of composite nanoshells were obtained both before and after coating with the hydrogel copolymer for experimental consistency.A Cary 50 Scan UV-vis optical spectrometer (Varian) with Cary Win UV software was employed to characterize the optical properties of the THPC gold seed-attached SiOThe DLS instrument  was operated with a light source wavelength of 514.5 nm and a fixed scattering angle of 90° for the hydrodynamic diameter measurement of the particles. After measuring 5 times for a duration of 100 s, the average hydrodynamic diameter was determined.m-SiO2 interlayer along with a hydrogel copolymer outerlayer, designed for enhanced drug delivery. The core gold nanoshells were prepared with a diameter of ~140 nm and an extinction maximum at 800 nm. We directly coated particles with a silica layer (~50 nm), which was then etched into a porous structure. The hydrogel overlayer was grown through emulsion polymerization to give a shell thickness of ~150 nm. The morphology, elemental composition, and physical properties of the composite particles were characterized by SEM, EDX, TEM, DLS, and UV-vis spectroscopy. Collectively, the data support the formation of distinct hydrogel-encapsulated m-SiO2-coated gold nanoshells. Through DLS analysis, the hydrodynamic diameters of the hydrogel-coated composite nanoparticles were shown to reproducibly decrease with increasing temperature over the range of 25 °C to 40 °C. The temperature-responsive behavior of the GNS@m-SiO2@hydrogel was demonstrated through DLS measurements, showing a reversible change of the particle diameter with changing temperature. The surface plasmon resonance of the GNS@m-SiO2@hydrogel exhibited an extinction maximum at ~810 nm, which falls in an important spectral range for biomedical applications. The NIR-responsive behavior of the GNS@m-SiO2@hydrogel was demonstrated by experiments involving a NIR laser (wavelength = 810 nm), showing that these composite particles, upon excitation by the laser, undergo an increase in temperature and respond accordingly.This research has revealed a method for the reproducible preparation of gold nanoshells and their encapsulation with a biocompatible m-SiO2@hydrogel to light at NIR wavelengths and to changes in temperature are completely consistent with the targeted objectives for their use as drug-delivery vehicles. The GNS@m-SiO2@hydrogel showed an improved loading capacity as compared to the particles prepared without a porous silica shell and an increase in the numbers of small molecules subsequently released. The rate and amount of the released molecules from these composite NPs could be regulated by adjusting the temperature and the exposure to the NIR laser beam. Given this combination of properties, our GNS@m-SiO2@hydrogel NPs offer considerable promise for use in targeted photo-initiated drug delivery.Finally, our fabrication method can be applied for use on other nanoparticle systems including various composite structures. The response of the hydrogel-encapsulated GNS@


Five among them: miR-204-5p, miR125b-5p, miR-125a-5p, miR320a, miR-99b—were upregulated in T2D+ patients with obesity, while only two miRs, miR-23b-3p, and miR197-3p, were increased in T2D– patients with obesity. These seven miRs target two groups of genes: matrix metalloproteinases and TGFβ signal pathway genes. According to the results of transcriptome analysis, the main difference between T2D+ and T2D– patients with obesity was in adipogenesis and fibrosis regulation by matrix metalloproteinases and SMAD4-RUNX2 signal cascade. Based on the data about transcription profiles of both groups, we suggested that the process of fibrosis in T2D+ patients with obesity is more pronounced than in T2D– patients with obesity.Obesity is a high-risk factor for such comorbidities as cardiovascular disease, several types of cancer, and type 2 diabetes; however not all individuals with obesity have such complications. Approximately 20% of individuals with obesity are metabolically healthy. This study focused on differences between obese individuals with and without type 2 diabetes  on the transcriptome level. Subjects included were 35 T2D– patients with obesity and 35 T2D+ patients with obesity with the same body mass index (BMI). The study was based on the transcription analysis of mRNA and microRNAs (miRs) by RNAseq. In the first step, we performed RNAseq of miRs, in the second step, we analyzed only those mRNA, which appeared targets for significant miRs from the first step. All RNAseq results were validated by qPCR. There were seven miRs differently expressed with adjusted  Other important characteristics of obesity are the percentage and content of adipose tissue. The adipose tissue is composed of white adipose tissue (WAT) and the brown adipose tissue (BAT), which have different morphology, distribution, gene expression, and function  . MiRs arThe aim of the present study was to identify differences in miR and mRNA expression between T2D+ and T2D– patients with obesity. We started from RNAseq analysis of miRs; next, we analyzed only those mRNA, which appeared targets for significant miRs from the first step.The study was approved by the Endocrinology Research Center ethics committee . Written informed consent was obtained from all the volunteers.2). Subjects <18 years old and with any other type of diabetes, pregnancy, or cancer were excluded from the study. T2D+ patients were diagnosed before including in the project on the basis of the Endocrinology Research Center. Obese T2D+ individuals were prescribed diabetes medications: 22.8% of patients received monotherapy, 42.9%- two-component therapy, 34.3%- three-component (Seventy patients (35 in T2D– group and 35 in T2D+ group) participated. All patients had a long duration (>15 years) of morbid obesity  using standard kits offered by the manufacturer. Glycated hemoglobin (HbA1c) was assessed with high-performance liquid chromatography . The lipid profile was measured by Architect c4000 analyzer .Insulin resistance was measured by two methods: the HOMA-IR calculator and the hyperinsulinemic-euglycemic clamp test.The HOMA-IR was calculated by the formulae:where FI = fasting insulin μIU /ml. And G = fasting glucose (mmol/l).The classic DeFronzo hyperinsulinemic-euglycemic clamp test was used to assess insulin resistance (IR) . InsulinThe tissue samples from patients were immediately placed in lysis buffer  and homogenized by a TissueLyser LT . Total RNA isolation from adipose tissue was carried out by an Rneasy Lipid Tissue Mini Kit  on the automatic QIAcube station according to the manufacturer protocol. To prevent degradation, we added 1 unit of RiboLock Rnase Inhibitor  per 1 μL of RNA solution. The concentration of total RNA in the aqueous solution was evaluated on a NanoVue Plus spectrophotometer .MiRs expression was then analyzed by sequencing on Illumina NextSeq 500 . The libraries were prepared by the Illumina TruSeq Small RNA Library Prep Kit following the manufacturer standard protocols. Strand-specific sequencing was performed for a total of 72 samples (31 samples from T2D– patients with obesity and 31 samples from T2D+ patients with obesity). The bioinformatics processing was as follows: adaptors were trimmed with Cutadapt; the resulting FASTQ files were then mapped onto the human genome (GRCh37 assembly) with bowtie2. FastQC was used as a tool to visualize different quality control measurements.http://microrna.sanger.ac.uk/sequences/) in SeqBuster. The resulting count data were analyzed in DESeq2 to obtain differentially expressed miRNAs. Only log2-fold changes with an adjusted p-value of 0.10 were considered significant. We used TargetScan, Diana-TarBase v8, and mirPath v.3 for sequence-based target prediction.For each sample, sequences were annotated using human pre-miRNA and mature miRNA databases provided by the miRBase . The bioinformatics processing was as follows: adaptors were trimmed with Cutadapt; the resulting FASTQ files were then mapped onto the human genome (GRCh37 assembly) using STAR. HTSeq was used to count the number of reads, and resulting count data were analyzed in edgeR to obtain differentially expressed mRNAs.The miRNet platform was used to integrate miRs and their target genes.2-fold changes with an adjusted p-value of 0.10 were considered significant.Pheatmap (R package) was used for the heatmap diagram; only logReverse transcription-quantitative PCR (RT-qPCR) of miRs in 70 samples was carried out by a TaqMan Advanced miRNA cDNA Synthesis Kit  and TaqMan Advanced miRNA Assays , in 96-well plates on the StepOnePlus PCR System , according to the manufacturer protocols. All samples were normalized to hsa-miR-191 levels and spike-in control cel-miR-39-3p. RT-qPCR assays were performed to validate the DESeq2 differential expression results for miRNAs with mean of normalized counts > 100.A two-step qRT-PCR of mRNA in 70 samples was carried out using a High-Capacity RNA-to-cDNA Kit  and Custom TaqMan Array 48 Plus plates , in 96-well plates on the StepOnePlus PCR System , according to the manufacturer protocol. All samples were normalized to GUSB and levels of GAPDH, HPRT served as secondary internal controls.P value < 0.05 was considered statistically significant. In order to account for multiple comparisons, a correction for the false discovery rate  was calculated using the Benjamini-Hochberg adjustment.Data analysis was performed by SDS software (version 2.3. Applied Biosystems). A According to the design of the project, subjects had no significant difference in BMI, but a comparison of HOMA-IR and M-index revealed substantial differences between the two groups of patients. Patients characteristics are shown in p-value ≤ 0.05 and fold-change ≥ 2.0 After the removal of low-quality reads and adaptor sequences, the mean yield of clean reads was 8.73 million (range: 6.13–15.79 million). Length distribution analysis of all samples revealed an expected peak at 20–24 nts, consistent with the size of miRs, while there was another one peak at 32–33 nts. There were 345–679 known miRs annotated in samples, most of the miRs were detected in all samples. All sample-specific miRs were lowly expressed (count <10), suggesting that these are of limited biological meaning.The miRNA-seq generated an average of 9.2 million single-end reads per sample, a total of 207 mRNA genes appeared to be DE with a significance threshold of adjusted p-value < 0.1, which were confirmed by qPCR  and TGFβ/BMP signaling components . As expected, target genes had decreased levels of expression.Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases capable of destroying the components of the extracellular matrix, as well as activating many biologically active molecules  . SubstraVarious studies reported varied results concerning MMPs expression profile, which can be related to the high heterogeneity of WAT , 28. ButIn our study, the MMPs expression was higher in T2D– patients with obesity, and such results are consistent with the study of Kosmala et al., who observed increased MMP-2 and MMP-9 activity in obese non-diabetic patients; herewith such processes were not observed in obese diabetic patients . We suppSMAD4 and RUNX2, which are components of TGFβ signals, became the second group of genes that increased their expression in adipose tissue of T2D– patients with obesity. High level of the expression of these genes corresponded with decreased expression of mir-204-5p, mir-125b-5p, mir-125a-5p. The essence of activation of both TGFβ- and VMR-dependent signaling cascades is the activation of corresponding receptors with subsequent phosphorylation of SMAD proteins of different types . For TGFHowever, according to Human Protein Atlas, neither SMAD4 nor RUNX2 is detected as protein products in adipose tissue . mRNA ofThe simultaneous increase of MMPs and SMAD4-RUNX2 genes in T2D– patients with obesity is consequent, as MMP9 presents a direct target of RUNX2. It has been shown that overexpression of RUNX2 significantly increases the endogenous levels of MMP9 by directly binding to a functional Runx2 regulatory element  MMPs genThe second limitation lies in cell heterogeneity of WAT. It consists not only of adipocytes but also fibroblasts, endothelial cells, immune cells, and preadipocytes . MoreoveThe present study is cross-sectional and do not give exact evidence of causality of obesity complications, however, it provides potentials target for drug development against T2D in individuals with obesity. Comprehension of the transcriptome difference between T2D+ and T2D– patients with obesity will allow to select the groups with high and low risk, and to determine the cohort of individuals, requiring the most intensive observation and treatment.The question of differences in pathological processes between individuals with obesity with the same BMI remains open. There is a growing body of evidence, that the key role in these cases belongs to miRs, as an important regulator of adipocyte metabolism. Based on the differential expression of specific miRs and mRNAs, we hypothesize that the main difference between T2D+ and T2D– patients with obesity consisted of adipogenesis regulation and prevailing of fibrosis processes in T2D+ patients with obesity. For further investigation of this problem, it is necessary to extend the patients cohorts and to engage cell and animal models.PRJNA565427 (https://www.ncbi.nlm.nih.gov/sra/PRJNA565427).The datasets generated for this study can be found in the Sequence Read Archive (SRA) The studies involving human participants were reviewed and approved by the Endocrinology Research Center ethics committee , Endocrinology Research Center, Moscow, Russia. The patients/participants provided their written informed consent to participate in this study.AN, DK, ES, ISk, OB, AP, MM, AK, AY, VF, and YY: data collection, analysis, and interpretation. AN, ES, OB, ISt, and MS: study conception and design. AN and MS have interpreted the data and reviewing manuscript. ISk and OB drafting and critical revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The characterization and properties of Z-LWACFs were investigated by nitrogen adsorption/desorption, X-ray photoelectron spectroscopy (XPS), methylene blue (MB) and iodine adsorption. Two activation process methods were employed to prepare Z-LWACF and contrasted with others fibers. The results showed that the Z-LWACF obtained by one-step ZnCl2 activation present higher yields and specific surface area than others fibers. Besides, the change of MB adsorption value at different impregnation ratios was consistent with pore structure distribution above 1.5 nm pore size, indicating that larger micropores (1.5 to 2 nm) and mesopores played a major role in the MB adsorption by Z-LWACF. The kinetics of MB adsorption process was found to follow the pseudo-second-order kinetic model and the adsorption rate was controlled by chemisorption. It was also found that MB adsroption by Z-LWACF belonged to monolayer adsorption and Z-LWACF was easy to adsorb MB.Activated carbon fiber was prepared from liquefied wood by chemical activation with ZnCl Presently, several environmental problems, including the greenhouse effect, water pollution and air pollution have attracted increasing attention from governments and researchers all over the world. Dyes, one of water pollutants, have been widely used in many industries . MethyleVarious methods such as adsorption, advanced oxidation, biosorption and membrane filtration have been extensively applied in the treatment of contaminated water . From th2 activation and found the iodine adsorption amount and yield rate of WACFs were 779.22 mg/g and 51.48% at 900 °C. Adsorbate substance can be used to characterize different pore size structures based on their particle size. Due to the large molecular linear size of MB, it is generally used to characterize the pore structure with a pore size greater than 1.5 nm on the surface of the adsorbent or to evaluate the decolorization ability of ACF. In addition, Iodine molecules are spherical molecules with a diameter of about 0.54 nm, and the area occupied by adsorption is about 0.39 nm2 or more. Therefore, iodine adsorption is usually used for the micropore characterization of porous materials.Many raw materials, including polyacrylonitrile fiber , phenoli2 activation (Z-LWACF) under various impregnation ratios were measured. The effect of activation conditions on the microporous adsorption performance and decolorization ability of Z-LWACF was investigated. Furthermore, the adsorption equilibrium, kinetics and adsorption isotherm of MB on Z-LWACF were determined and correlated with isotherm equations.In this work, the activation process technology of liquefied wood carbon fiber by one-step method and two-step method were studied and evaluated. The iodine and methylene blue adsorption value of activated carbon fibers prepared from liquefied wood by ZnClChinese fir (Cunninghamia lanceolate), sourcing from Fujian, China, was used as a raw material. Phosphoric acid, phenol, hydrochloric acid, hexamethyleneteramine, formaldehyde, sodium thiosulfate, disodium hydrogen phosphate, monopotassium phosphate, copper sulfate pentahydrate and zinc chloride were purchased from Beijing Chemical Works . Soluble starch was purchased from Xilong Chemical Co. Ltd. . Potassium iodide and iodine was purchase from Guangdong Guanghua Sc-tech Co. Ltd. . Methylene blue was purchase from Tianjin Jinke Fine Chemicals Co. Ltd.  All the chemicals were of reagent grade and used without further purification.Chinese fir through a series of process including liquefaction, melt-spinning and curing, according to our previous study by the authors [2 aqueous solution was brought into contact with about 2 g of the material. The amount of activating agent present in such a solution was that corresponding to a given ZnCl2: precursor fibers ratio (3~6 by weight). Then, ZnCl2-impregneated fibers were placed in a horizontal transparent tube furnace  and heated from room temperature to 700 °C in a N2 atmosphere. The heated rate was 4 °C/min, and the holding time at 700 °C was 1 h. The produced sample were washed in 1 mol/L HCl, rinsed several times in distilled water until the pH of water became neutral. Finally, the fibers were dried in an oven at 103 ± 2 °C for 24 h to obtain the ACFs. The final yield of ACFs was determined as the weight ratio of ACFs to precursor fibers. The as-prepared ACFs were denoted as Z-LWACF-R, where R  represented the impregnation ratio. In addition, two activation methods were employed to prepare ACFs (Y) is defined as the ratio of the weight of Z-LWACFs to that of the precursor fibers.mp is the mass of the liquefied wood precursor fiber and mz is the mass of Z-LWACF after activation, washing, and drying.Precursor fibers were prepared from  authors . In the are ACFs . These A2 adsorption-desorption isotherms at −196 °C . Before analysis, all the ACFs were degassed at 300 °C for 3 h. The specific surface area (SBET) of prepared ACFs was estimated by BET method. The total volume  was estimated to be the liquid volumes of N2 at high relative pressure (P/P0 = 0.995). The micropore volume (Vmicro) and mesopore volume (Vmeso) were determined by t-plot analysis method and BJH method. The pore size distribution (PSD) of the prepared ACFs have been calculated by the density functional theory (DFT) method.XPS analysis was performed using a spectrophotometer  with a monochromated AlKα X-ray source . The survey scans were collected from a binding energy ranging from 0 to 1350 eV. Pore structure parameters of those prepared ACFs were measured by NCt) of MB was measured according to the method just described above, and finally the adsorption value (Qt) of Z-LWACF on different MB concentrations was calculated using the following equation:C0 is the initial MB concentration, Ct is remaining concentration at adsorption time, V is the volume of aqueous solution and m is the mass of Z-LWACF.The MB adsorption value of Z-LWACFs prepared under different activation conditions were determined according to the Chinese national standard GB/T 12496.10-1999 . The preA) of Z-LWACF was calculated by the following equation:C1 is the molarity of iodine standard solution, C2 is the molarity of sodium thiosulfate standard solution, V is the volume of sodium thiosulfate solution, M is the mass of Z-LWACF, D is the correction coefficient.The iodine adsorption value of Z-LWACF prepared under different activation conditions was determined according to China national standard GB/T 12496.8-1999 [2 after impregnation. The carbonization and activation have simultaneously occurred in the precursor fibers. Under low temperature, zinc chloride has a protective effect on the carbon body so that the precursor fibers can form a relatively stable condensed carbon structure and reduce the incidence of by-products. Then pores were produced and grown in the carbon fibers under the activation of ZnCl2, leading to a decrease of yield and an increase of specific surface area. Therefore, one step method would be more suitable for producing ACFs from liquefied wood by ZnCl2 activation. Furthermore, it is obvious from the data shown in 2 activation present higher yields and BET surface area than those prepared by others methods [2 would show good adsorption properties.The yields and specific surface area of ZACF prepared from liquefied wood by one and two step methods and others ACFs were shown in  methods ,27,28,29The XPS survey scan spectra of ZnCl2-activated ACFs with various impregnation ratios are shown in The curve-fitting results of C1s peak of Z-LWACFs prepared under different impregnation ratios are shown in 2 impregnation ratios and the pore size distribution of pore structure in the range of 1.5 nm to 25 nm are shown in Since the molecular linear size of methylene blue is large and the pore size of the adsorbent should be greater than 1.5 nm, MB is often used to characterize the decolorization ability of materials and the development degree of pore structure above 1.5 nm of porous materials. The MB adsorption value of ACFs prepared at different ZnClIodine adsorption mainly occurs on the surface of adsorbents with a pore radius of about 0.8 nm to 1.0 nm. It is usually used to reflect the adsorption capacity of activated carbon fibers for small molecules to a certain degree . The iodThe curve of the adsorption amount of Z-LWACF-6 in MB solution which under the initial concentrations of 120 mg/L, 240 mg/L, 360 mg/L, 480 mg/L, and 600 mg/L over time is shown in Qe and Qt (mg/g) represent the amounts of MB adsorption at equilibrium state and any time (h), respectively. k1 (h−1) is the rate constant of the pseudo-first-order model and k2 (g·mg−1·h−1) is the pseudo-second-order rate constant.Adsorption kinetic analysis are thoroughly investigated at the condition of 25 °C and initial pH value (6.3–6.5), which were suitable for the adsorption of cationic dyes like MB .The adsoR2 of the pseudo-first-order kinetics of Z-LWACF-6 adsorbing MB at different initial concentrations was 0.9078~0.9505, and the fitting effect was poor. When the initial concentration increased, the theoretical equilibrium adsorption amount increased gradually. It implied that the equilibrium adsorption amount was closely related to the initial concentration of MB. However, the theoretical value and the actual equilibrium adsorption amounts were quite different, indicating that the pseudo-first-order model could not accurately describe the whole process of MB adsorption onto Z-LWACF-6. According to previous literature [The pseudo-first-order kinetic model for the adsorption of MB onto Z-LWACF-6 and the equation parameters are shown in terature , this wok2 gradually decreased from 0.4489 to 0.0098. It was obvious that the theoretical equilibrium adsorption calculated by pseudo-secondary kinetics was close to the experimentally measured equilibrium adsorption at different initial concentrations. In addition, the correlation coefficient of pseudo-secondary dynamics fitting was between 0.9998~1 and the fitting results were better than pseudo-first-order kinetics. This indicated that the pseudo-second-order kinetics was more suitable for describing the process of Z-LWACFs for MB adsorption [sorption , includiKL (L/mg) is the Langmuir constants involved in adsorption rate, and Qm (mg/g) is adsorption amount. KF and 1/n are Freundlich constant and empirical constant.Langmuir and Freundlich models were adopted to analysis the isotherm data of MB adsorption onto Z-LWACFs. These two models can be expressed as follows:R2 (0.9822) of the Langmuir adsorption isotherms was close to 1 and better than Freundlich model (0.9682), indicating the good application of Langmuir model [n calculated by Equation (7) is frequently used to characterize the adsorption intension. When 1/n is in the range of 0.1 to 0.5, it indicates that the adsorption is easy occurred. From the table, the 1/n value was 0.22, indicating that MB is easy to be adsorbed by Z-LWACFs.The experimental data were fitted using Equations (6) and (7). The Langmuir and Freundlich isotherms are displayed in ir model . FurtherThe trend of MB adsorption amount of Z-LWACF at different impregnation ratios was consistent with pore structure distribution above 1.5 nm pore size, indicating that larger micropores (1.5 nm to 2 nm) and mesopores played a major role in the MB adsorption by Z-LWACF. The iodine adsorption capacity of Z-LWACF is consistent with the change of pore structure at pore diameters of 0.8 nm to 1.5 nm and 2 nm to 4 nm. Under different initial concentrations, the adsorption amount of Z-LWACF increased rapidly with the increase of adsorption time. With the increase of initial concentration, the equilibrium adsorption capacity of Z-LWACF increased gradually. The fitting results show that the pseudo-second-orde kinetic model is more suitable for describing the adsorption process of Z-LWACF to MB, indicating that the rate of MB adsorption is controlled by chemisorption. The results of adsorption isotherms show that the adsorption of MB by Z-LWACF belongs to monolayer adsorption and Z-LWACF is easy to adsorb MB.


Purpose: As a Chinese medicinal herb, Desmodium styracifolium (Osb.) Merr (DS) has been applied clinically to alleviate crystal-induced kidney injuries, but its effective components and their specific mechanisms still need further exploration. This research first combined the methods of network pharmacology and proteomics to explore the therapeutic protein targets of DS on oxalate crystal-induced kidney injuries to provide a reference for relevant clinical use.Methods: Oxalate-induced kidney injury mouse, rat, and HK-2 cell models were established. Proteins differentially expressed between the oxalate and control groups were respectively screened using iTRAQ combined with MALDI-TOF-MS. The common differential proteins of the three models were further analyzed by molecular docking with DS compounds to acquire differential targets. The inverse docking targets of DS were predicted through the platform of PharmMapper. The protein–protein interaction (PPI) relationship between the inverse docking targets and the differential proteins was established by STRING. Potential targets were further validated by western blot based on a mouse model with DS treatment. The effects of constituent compounds, including luteolin, apigenin, and genistein, were investigated based on an oxalate-stimulated HK-2 cell model.Results: Thirty-six common differentially expressed proteins were identified by proteomic analysis. According to previous research, the 3D structures of 15 major constituents of DS were acquired. Nineteen differential targets, including cathepsin D (CTSD), were found using molecular docking, and the component-differential target network was established. Inverse-docking targets including p38 MAPK and CDK-2 were found, and the network of component-reverse docking target was established. Through PPI analysis, 17 inverse-docking targets were linked to differential proteins. The combined network of component-inverse docking target-differential proteins was then constructed. The expressions of CTSD, p-p38 MAPK, and p-CDK-2 were shown to be increased in the oxalate group and decreased in kidney tissue by the DS treatment. Luteolin, apigenin, and genistein could protect oxalate-stimulated tubular cells as active components of DS.Conclusion: The potential targets including the CTSD, p38 MAPK, and CDK2 of DS in oxalate-induced kidney injuries and the active components  of DS were successfully identified in this study by combining proteomics analysis, network pharmacology prediction, and experimental validation. The worldwide prevalence of kidney stone disease, also known as nephrolithiasis, is ~10% with a continuous increase in recent years  Merr (DS) is one of the most popular traditional Chinese herbs, and its dried aerial part has been extensively applied in the clinical therapy of renal stones. DS is confirmed to reduce CaOx deposition in the kidneys  and maintained in DMEM-F12 supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in 5% COAll animal studies were performed in accordance with the National Institutes of Health (NIH) guide for the Care and Use of Laboratory Animals. The experimental procedures were approved by the Ethical Committee for the Experimental Use of Animals at Second Military Medical University .Sixteen wild-type male C57BL/6 mice (7–8 weeks old) were purchased from Shanghai SLAC Animal Co., Ltd. . All mice had free access to drinking water and regular chow every day and were kept under a controlled 12 h light/dark cycle at 20–25°C with the relative humidity at 55–65%. After conditioned housing for 1 week, 16 mice were equally divided into the control group and the oxalate model group, with eight mice each. The oxalate group was administered glyoxylate (100 mg/kg/day) by daily intra-abdominal injections for 7 consecutive days, and the control group was intra-abdominally injected daily with normal-volume saline (20 mL/kg/day) per day. On day seven after the administration of glyoxalate, kidney samples were collected, and the cortex and medulla junction tissue were dissected to be further analyzed.ad libitum. After 1 week of habituation, the rats were randomized according to their weight into the control and oxalate model groups, with eight rats in each group. A model of oxalate kidney stones was established by the administration of food, which was supplemented with 5 g/kg dose of hydroxyproline per day; the control group received the same volume of 0.5% CMC-Na. No animals died or showed severe clinical symptoms before reaching the 28 days of treatment. At the end of 28 days, the rats were sacrificed and both kidneys of the rats were excised. After removing the capsule and pelvis, kidney samples were collected, and the cortex and medulla junction tissues were dissected for further analysis.Sixteen male Sprague-Dawley rats (120–130 g) at the age of 5 weeks were purchased from Shanghai SLAC Laboratory Animal Co., Ltd . Three rats per cage were maintained under standard laboratory conditions  with aseptic food and tap water For protein extraction, cells and the cortex and medulla junction tissue from rats or mice were respectively homogenized and lysed in lysis buffer . Then, the homogenate was centrifuged at 13,000 r/min for 10 min at 4°C, and the supernatant, which contained proteins, was recovered. The protein concentrations in each sample were measured using a BCA kit (Thermo Fisher Scientific) according to the manufacturer's protocol.2O to 30% of the total reaction volume to inactivate the iTRAQ regents.After the concentration determination, equal aliquots from protein samples in the same group were mixed together. A 120 μL portion of the pooled protein sample from each group was transferred into one tube and reduced with TCEP, alkylated with MMTS, and digested with trypsin at room temperature according to the iTRAQ (isobaric tags for relative and absolute quantitation) reagent reference guide . The obtained tryptic peptides from the control and oxalate groups were respectively labeled with iTRAQ reagents with different tags for 2 h at room temperature and then terminated by adding HThe total proteins of mice and HK-2 cells were analyzed based on Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The dried pooled peptides were then reconstituted in A phase  for high-pH reverse-phase liquid chromatography (RPLC) separation, using a Shimadzu LC−20AB HPLC system with an Ultremex SCX column (250 × 4.6 mm), with an 0.8 mL/min flow rate. Fractions were collected and combined into new fractions according to parameters. MALDI-TOF-MS was used for protein identification. The combined fractions were mixed with MALDI matrix  containing 10 mM ammonium phosphate). The matrix was delivered with a PHD200 infusion pump (Harvard Apparatus) at 0.8 L/min. Fractions were deposited at 30-s intervals on a stainless steel MALDI target plate . The MS was operated in positive ion mode across the mass range of 800–1250 m/z using a 5800 TOF/TOF spectrometer (Applied Biosystems/MSD Sciex). The scan of MS/MS was completed in a data-dependent manner, and the most abundant 15 peaks were selected in per spot with a minimum signal-to-noise ratio of 40. The atmosphere was used as collision gas with collision energy of 1 kV. The proteins of rats were analyzed using LC-MS/MS analysis, and the detailed method is described in the Supplementary Material. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD009496.http://www.uniprot.org/). For data integration and validation, the search results were input into Scaffold Q+ software . The peptide and protein false-discovery rate (FDR) were both set to ≤1%, and protein identifications were accepted if they contained at least two identified peptides and at >99% probability. For quantitation analysis, Scaffold Q+ was used to quantify the isobaric tag peptide and protein identifications. Mann-Whitney U-tests for each group were calculated using SPSS 20.0, and p < 0.05 was considered statistically significant. In this experiment, proteins with a concentration ratio  > 2.0 and < 0.5 were considered the significantly differential expression proteins.All MS/MS data were acquired with a data converter of AB sciexms (version 1.3). The acquired peak-lists of all MS/MS spectra were searched with Mascot  against the corresponding mouse, rat or human database downloaded from Uniprot Knowledgebase (https://pubchem.ncbi.nlm.nih.gov) to get the 3D molecular structure of each compound saved in “SDF” file format.Information on the main compounds in DS was acquired according to previous research  as potential targets for docking. The whole work of docking was conducted using the commercial software Discovery Studio 2.5 (http://www.accelrys.com). First, the X-ray crystal structures of protein targets were preprocessed. Hydrogen was added to the model, and its orientation was optimized using the CHARMm force field energy minimization while all non-hydrogen atoms were not allowed to move. Each protein was defined as a receptor, and the proteins' active sites were found from the receptor cavities using the Discovery Studio tool. Then, the docking protocol was performed to show the interaction of components in DS with the differential proteins using LibDock. As Libdock can provide 10–100 predicted dockscores from different docking poses for each compound in a binding pocket of a protein, we only considered the best dockscore. The cutoff value of the dockscore for differential targets was set as 80 in this protocol.The differential proteins acquired from proteomic analysis were considered potential proteins related to crystal-induced kidney injuries. The human structures of these differential proteins were collected from the protein data bank (PDB) , which is an updated integrated pharmacophore matching platform for the identification of potential targets with inverse-docking approaches , which is a database of known and forecasted PPIs, with the species limited to “Homo sapiens” and a confidence score >0.4. Each node represents proteins produced by a single protein-coding gene locus.To explore the indirect effect of DS on the differential proteins, the PPI between potential inverse-docking targets from PharmMapper and the differential proteins were analyzed by String (http://www.cytoscape.org/), which is an open-source software project for integrating biomolecular interaction networks with high-throughput expression data into a unified conceptual framework.To facilitate the scientific interpretation of complex relationships between active components of DS and nephrolithiasis-related protein targets, the network was generated by Cytoscape . The dried aerial part of the DS plant was pulverized to powder by a disintegrator. The powder (250 g) was extracted with 1 L of 75% ethanol for 2 h by an ultrasonic method at room temperature. The extraction solution was filtrated followed by concentration using a rotary evaporator at 55°C to dryness. The yield of the ethanol extraction was 12.7% (w/w). The extract powder was dissolved in saline solution to obtain a concentration of 50 mg/ml for the following experiments.Twenty-four wild-type male C57BL/6 mice (7–8 weeks old) were purchased from the Cavens . After conditional housing for 1 week, these mice were randomly divided into four experimental groups including a control group, an oxalate group, a DS low-dose group, and a DS high-dose group with six mice in each group. To establish the crystal renal injury model, the mice were intraperitoneally injected with glyoxylate at a dosage of 100 mg/kg once daily for 7 days, except for the control group, which was injected with saline. After each glyoxylate injection, the mice in the two DS groups were respectively given the DS extract at a low dose of 10 mL/kg  and a high dose of 20 ml/kg by gastric perfusion. Mice from the control and oxalate groups were i.g. administered saline.On day 7 after the administration of glyoxalate, blood was collected from the orbital sinus, and the serum sample was separated by centrifugation at 4,000 rpm at 4°C for 5 min. The supernatant was aliquoted into sterile tubes and frozen at −80°C. After urine and blood collection, bilateral kidney tissue samples from 24 mice were harvested. Sections of the right kidneys were fixed in neutral buffered formalin (10%) for further analysis. The left kidneys were dissected and frozen at −80°C for future use.https://imagej.nih.gov/ij/). Some remaining sections were colored with the hematoxylin-eosin for evaluation under an optical microscope.The fixed kidney samples were further embedded in paraffin and sectioned at a thickness of 3 μm. After staining with the Von Kossa method according to the instructions of the commercial kit , images of randomly selected areas from each section were collected by a light microscope. Twenty views were gathered from each group for semi-quantitative examination by Image J software  and 8-hydroxydeoxyguanosine (8-OHdG) (H165) in kidney tissue and the urinary ratio of calcium to creatinine (Ca/Cr) (C011-1) were respectively measured using a corresponding commercial kit . The concentration of the serum excretion of kidney injury molecule-1 (KIM-1) was tested using an ELISA kit .2. As previously described, oxalate-induced kidney injury models of HK-2 cells were established with NaOx at 1 mM for 12 h as the experimental conditions. Luteolin, apigenin, and genistein were obtained from MCE  and were resuspended in DMSO to be used at indicated concentrations. HK-2 cells in treated groups were co-incubated for 12 h with various concentrations of luteolin, apigenin, or genistein with NaOx.HK-2 cells were maintained in DMEM-F12 supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg /mL streptomycin at 37°C in 5% COCell proliferation was monitored using a Cell Counting Kit-8 (CCK-8) . In brief, HK-2 cells were suspended in medium with or without NaOx stimulation and compound treatment and then plated in 96-well plates at a 2000 cells/well concentration. Cell proliferation was measured after adding CCK-8 reagent 1 h at a 450 nm absorbance.CTΔΔ method.Total RNA was acquired from HK-2 cells using TRIzol reagent . The absorbances at 260 and 280 nm were measured to confirm the quantity and purity of total RNA. cDNA was synthesized from total RNA using reverse transcriptase M-MLV (RNase H-) . SYBR Green Master Mix  was used to measure the relative expression levels of mRNA. All the primers were designed and synthesized by Sangon, Shanghai, China, and the sequences of primers are listed in Supplementary Table Acquired cells from three repeated experiments and corticomedullary tissues from 24 mice were homogenized in lysis buffer  containing protease inhibitor and phosphatase inhibitor. The lysates were centrifuged at 12,000 rpm for 5 min at 4°C and the supernatant was collected. The protein concentration of mixed lysates was determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of total protein were subjected to SDS-PAGE gel for separation and transferred onto a nitrocellulose membrane . After blocking, the membrane was incubated with rabbit polyclonal anti-CTSD , anti-phospho-CDK2 (Thr160) , and anti-phospho-MAPK 14(Thr180/Tyr182)  antibodies at 4°C overnight. After washing with TBST, the membrane was incubated with a fluorescence-conjugated secondary anti-rabbit antibody  for 60 min at room temperature. The signals were visualized using the Odyssey Infrared Imaging System  and quantitatively analyzed by normalizing them to β-actin using the Image J software.U-test was further employed for post-hoc comparisons. The significance level was set at p < 0.05.Data are expressed as mean ± SEM and were plotted in histograms with GraphPad Prism 5.0. Statistical analysis was performed using a one-way analysis of variance (ANOVA) or the Kruskal-Wallis H test as appropriate to assess differences among groups. Bonferroni's test or the Mann-Whitney Supplementary Figure We obtained 20 X-ray crystal structures of differential proteins from PDB as potential targets for the docking analysis of the 15 main components in DS. The detailed docking results are listed in Supplementary Table DS may also indirectly affect these nephrolithiasis-related differential proteins through other protein targets. Information on more potential targets was obtained by performing inverse docking by PharmMapper. The details of the top 10 targets are described in Supplementary Table The differential proteins and the inverse-docking targets were imported into String together to be analyzed based on the PPI  is a member of the cyclin-dependent kinase family of Ser/Thr protein kinases and involved in the control of the cell cycle. The increased activity of CDK-2 has been discovered in adverse renal diseases, such as renal failure during sepsis  and the active components  of DS were successfully found based on this practical strategy.JH, WC, XD, and ZG conceived and designed the experiments. JH, HL, and SG performed the experiments. JH, HZ, and WL analyzed the data. WC, XD, and ZG contributed reagents, materials, analysis tools. JH, WC, and HL wrote the manuscript. All authors approved on the finally submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gluconacetobacter xylinus (G. xylinus) at the boundary of two non-mixed silicone oil layers. The fibrils of bacterial cellulose (BC) were produced at the interface of water and oil phases. Fibril layers effectively thickened layer-by-layer and eventually formed a shell structure. The size of the HSBC gel can be controlled by the volume of dropped cell suspension. For cell suspensions of 50 μL and 10 μL, HSBC gels of approximately 4.0 mm and 2.5 mm were obtained, respectively. The shell of the HSBC gel is the gelatinous membrane formed by well-organized fibril networks; they comprised type-I crystal structure of cellulose. Additionally, we studied release profile of the fluorescein isothiocyanate-dextran (FITC-Dex) and observed that it released rapidly from the HSBC gels compared to from the BC gels obtained by the static culture method. The release behavior from HSBC gel agreed satisfactorily with Higuchi model. Therefore, the shell of HSBC gel is surely a thin gelatinous membrane of BC, and would be useful as a drug release device.We developed a novel cultivating system for hollow-type spherical bacterial cellulose (HSBC) gel production without any molds or template. It consisted of floating aqueous medium droplet containing  Bacteriscovered  and has scovered . BC has scovered . In addiscovered , 2016.Gluconacetobacter xylinus; the most common method is static culture, which produces gelatinous cellulose mass at the interface between the air and the liquid culture media. In Indonesia and the Philippines, BC obtained by static culture is manufactured in large quantity as food called Nata de coco. However, its shape depends on that of the culture container.To obtain BC, several culture methods can be applied using Acetobacter xylinum strain NQ5 (ATCC 53582) produced isolated sphere-type cellulose  and culture medium, whereas, the oil phase can supply oxygen to support its culture. Preparation of spherical BC gels was predicted by culturing G. xylinus in medium droplets. Moreover, we hypothesized that it would be hollow-type spherical BC (as HSBC), that can have thin gelatinous membrane composed of cellulose networks, which would be produced at the interface of water and oil phases. Until now, to the best of our knowledge, there is no report about such HSBC gel and the culturing method. These HSBC gels are expected as a seamless capsule for drug delivery applications.Herein, we attempted to prepare the spherical BC gel by the formation of Water-in-Oil type droplets without any molds or template. The water phase contains 22.1TM, Nihon Pharmaceutical Co. Ltd.), 5.0 g BactoTM yeast extract (BD Biosciences), and 1.0 g magnesium sulfate heptahydrate  in 1000 mL MilliQ water. Fluorescein isothiocyanate-labeled dextran  was purchased from Merck Co. Silicone oils (KF-56 and KF-54) were obtained from Shin-Etsu Chemical Co., Ltd. Other reagents were purchased from Kanto Chemical Co. Inc., and used as-received.Hestrine-Schramm's medium (HS medium)  was used2.2G. xylinus (IFO13772) was cultured in the HS medium at 30 °C for 3 days. The cultured cell suspension was diluted with the same medium and dropped aseptically into 100 mL mixture silicone oils  and incubated at 30 °C for 14 days by keeping the droplets floating.The droplet size was controlled by the amount of drop (10–50 μL). The HSBC gel thus obtained was purified by soaking in a large quantity of distilled water for 1 day followed by washing in a 1% (w/v) aqueous solution of NaOH at room temperature for 1 day to remove the bacterial cell debris and alkali-soluble components. Then, it was washed several times with large quantity of distilled water and stored in distilled water at room temperature.G. xylinus were grown under static conditions in a glass test tube  at 30 °C for 14 days. First, the solution became turbid, and a BC gel appeared on the air-culture medium interface. The gel thickness increased gradually, reaching 10–20 mm after 14 days. Purification of conventional BC gel was carried out by the same method as the HSBC gel.As a comparative sample, the conventional BC gel obtained by the static culture method. 2.32 (scCO2) technique without disintegrating its microstructure , 50 mL pressure vessel, a gas pressure regulator , and a constant temperature water bath .After HSBC gel swelled in water, it was put in a large quantity of methanol and washed thoroughly and swelling solvent was changed from water to methanol completely. The gel was dried by supercritical COtructure . The dry2.4The microstructure of HSBC aerogels was observed using a field-emission scanning electron microscope (FE-SEM: Hitachi High-Technologies Corporation S-4500) with an acceleration voltage of 10 kV. For the pretreatment to FE-SEM observation, deposition of Pt-Pd was performed by ion sputtering (Hitachi High-Technologies Corporation E-1010).−1 in transmittance mode and 24 scans.To analyze the composition and the crystal structure of the obtained HSBC aerogels, wide angle X-ray diffraction (WAXD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) measurement were carried out. WAXD experiments were performed at 20 °C using an X-ray diffractometer . The Cu-Kα radiation  was generated at 40 kV and 200 mA. The sample was scanned at a rate of 3°/min between 10° and 40° in transmittance mode. The ATR-FTIR spectra were measured using a FTIR spectrophotometer (Perkin Elmer Spectrum One) equipped with universal ATR sampling accessory. All measurements were carried out with a nominal spectral resolution of 1 cm2.5To evaluate the drug release behavior from the obtained HSBC gel, FITC-Dex  was used as the model drug. The HSBC gel was immersed in a 1 mg/mL FITC-Dex aqueous solution and kept in a dark place at 4 °C for one day, and permeation of FITC-Dex into the HSBC gel was observed . The rel33.13 using the Baume hydrometer. Since silicone oil with the same density is not commercially available, we tried to adjust the density of silicone oil by two methods. The first method was to mix silicone oil and a miscible organic solvent having a density of >1 g/cm3 such as o-dichlorobenzene. In this method, however, although density control of the silicone oil was easy, the organic solvent inhibited the growth of G. xylinus, and hence, cellulose was not produced. The other was density adjustment by mixing silicone oils which have different specific gravities. Essentially, the two kinds of silicone oils, KF-56 (0.96 g/cm3) and KF-54 (1.07 g/cm3), are used in this method. Although these oils were immiscible and become cloudy when mixed , microfibril networks were produced layer by layer, thus forming a laminated structure that increased in thickness , 1163 cm−1 (C-O-C stretching), and 897 cm−1 (β-glucosidic linkage) that are specific to cellulose type I crystals  from within the HSBC gel (diameter: 3.50 mm) was studied by UV-Vis spectroscopy. For comparison, we used the conventional BC gel obtained from the static culture, which had almost the same surface area of the HSBC gel. The HSBC gel showed rapid release initially due to a thin gelatinous membrane as compared to the conventional BC gel, and the release rate gradually decreased (∞ was 22.6 μg.As described above, HSBC gel is like a seamless capsule. These walls comprise of cellulose fiber networks. To investigate its feasibility as a drug delivery device, the release behavior of FITC-Dex (Mecreased . At 3000The partition coefficient in the diffusion of dextran in the BC membrane is equal to 1 . The resThe release behavior was analyzed using the following Korsmeyer-Peppas model Eqs. t and M∞ are the absolute cumulative amount of drug released at time t and infinite time, respectively; k is a constant incorporating structural and geometric characteristics of the device, and n is the release exponent, indicative of the mechanism of drug release. From 2 values of Korsmeyer-Peppas model and Higuchi model are 0.926 and 0.950, respectively. Release behavior of model drug from HSBC gel agrees very well with Higuchi model (Here, Mhi model b. Such ghi model , and a m4G. xylinus at the boundary of two non-mixed silicone oil layers with different densities. The densities of the oils used were precisely controlled to enable the floating of droplets at the boundary for cultivating periods. The size of the HSBC gel can be controlled by the volume of dropped cell suspension. After cultivation, the HSBC gels were purified and dried by supercritical drying techniques to characterize the morphology of gels. Scanning electron microscopy proved the hollow structure and well-organized fibril networks; they comprised type-I crystal structure of the cellulose by careful measurement of infra-red spectroscopy. Thus, HSBC gels have thin gelatinous membrane composed of cellulose networks, which were produced at the interface of water and oil phases. The model drug was rapidly released, and the profile agrees satisfactorily with Higuchi model, which indicates that drug release mechanism followed Fickian diffusion for the thin films.We developed a cultivating system for HSBC gel production without any molds or template. It consisted of floating aqueous medium droplets containing Cellulose can modify various functional groups and functional polymers . ModifieToru Hoshi: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Takao Aoyagi: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Kazuyoshi Yamazaki, Yuki Sato, and Takaya Shida: Performed the experiments.JSPS KAKENHI Grant Number JP15K18726.This work was supported by The authors declare no conflict of interest.No additional information is available for this paper.